Bioprotection of Zea mays L. from aflatoxigenic Aspergillus flavus by Loigolactobacillus coryniformis BCH-4

Fungal infection causes deterioration, discoloration, and loss of nutritional values of food products. The use of lactic acid bacteria has diverse applications in agriculture to combat pathogens and to improve the nutritional values of cereal grains. The current research evaluated the potential of Loigolactobacillus coryniformis BCH-4 against aflatoxins producing toxigenic Aspergillus flavus strain. The cell free supernatant (CFS) of Loig. coryniformis was used for the protection of Zea mays L. treated with A. flavus. No fungal growth was observed even after seven days. The FT-IR spectrum of untreated (T1: without any treatment) and treated maize grains (T2: MRS broth + A. flavus; T3: CFS + A. flavus) showed variations in peak intensities of functional group regions of lipids, proteins, and carbohydrates. Total phenolics, flavonoid contents, and antioxidant activity of T3 were significantly improved in comparison with T1 and T2. Aflatoxins were not found in T3 while observed in T2 (AFB1 and AFB2 = 487 and 16 ng/g each). HPLC analysis of CFS showed the presence of chlorogenic acid, p-coumaric acid, 4-hydroxybenzoic acid, caffeic acid, sinapic acid, salicylic acid, and benzoic acid. The presence of these acids in the CFS of Loig. coryniformis cumulatively increased the antioxidant contents and activity of T3 treated maize grains. Besides, CFS of Loig. coryniformis was passed through various treatments (heat, neutral pH, proteolytic enzymes and catalase), to observe its stability. It suggested that the inhibitory potential of CFS against A. flavus was due to the presence of organic acids, proteinaceous compounds and hydrogen peroxide. Conclusively, Loig. coryniformis BCH-4 could be used as a good bioprotecting agent for Zea mays L. by improving its nutritional and antioxidant contents.

Introduction flavonoid contents, and antioxidant activity. High-performance liquid chromatography (HPLC) was performed to determine aflatoxins, present in untreated and treated maize grains. Another HPLC analysis was done to identify the presence of phenolic acids in cell free supernatant (CFS) of Loig. coryniformis BCH-4 which has been suggested to contribute in the bioprotection potential. The current study suggested the bioprotection of Zea mays L. against the toxicity of A. flavus along with improving the functional antioxidant contents of maize grains.

Microbial cultures, and growth conditions
Loigolactobacillus coryniformis BCH-4, previously named Lactobacillus coryniformis (KX388387), was isolated from fermented rice rinsed water [29]. The culture was grown in MRS medium for 48 h at 37˚C and preserved as glycerol stocks at -80˚C, for long term storage in 15% (v/v) glycerol. Pure culture of A. flavus, an aflatoxigenic filamentous fungus, was obtained from the Department of Bioinformatics and Biotechnology, Government College University Faisalabad, Pakistan (Accession No. MH179066) and was grown on Vogel's agar medium in Petri plate (100 × 17 mm diameter) for 48 h at 30˚C, and stored at 4˚C.

In vitro antifungal activity of Loig. coryniformis BCH-4 CFS against A. flavus
Agar well diffusion assay was performed for determining the antifungal potential of Loig. coryniformis BCH-4 CFS [31]. For this purpose, the A. flavus (10 6 spores/mL) culture was spread over solidified Vogel's agar medium Petri plate (100 × 17 mm diameter), using a sterile cotton swab. A 10 mg CFS was dissolved in 1 mL sterile distilled water and a total 60 μL of CFS was added in 8 mm diameter wells. Besides, a negative control was used, having a non-inoculated MRS broth medium instead of CFS. The Petri plates were evaluated for antifungal potential by measuring the zone of inhibition around the well after incubating for 48 h at 30˚C.

Effect of heat, pH, and proteolytic enzymes on antifungal activity of CFS
To determine the biochemical nature and stability of antifungal compounds, present in Loig. coryniformis, 10 mg lyophilized CFS was treated with proteinase K, pepsin, catalase, pH effect and heat toleration according to Oirdi et al. [33] with slight modifications. The sensitivity to heat treatment was investigated by subjecting the CFS to 100˚C for 20 min. To evaluate the pH effect, the CFS was neutralized with 1 M NaOH solution. The enzymatic effect on antifungal activity was determined by submitting 500 μL of CFS to 20 μL of enzymes catalase (10 mg/mL, phosphate buffer; pH 7), proteinase K (10 mg/mL phosphate buffer, pH 7), and pepsin (10 mg/ mL 1 M HCL, pH 2). The mixture was incubated at 30˚C for 3 h and the pH of CFS was readjusted to the initial pH value before each test. In addition, MRS broth and sterile distilled water both were used as control. All the treated CFS samples were analyzed using agar well-diffusion method against A. flavus as described above.

Bioprotection of maize grains
Preparation of A. flavus culture. A. flavus was grown in Vogel's broth medium in a 250 mL sterile flask at 30˚C for 7 days until sporulation occurred. The spore concentration was determined using a hemocytometer and adjusted to 10 6 spores per mL.
Bioprotective effect of CFS. The bioprotective effect of Loig. coryniformis BCH-4 CFS was determined on maize grains against A. flavus as reported by Nazareth et al. [34] with slight modifications. In short, 10 g of lyophilized CFS was dissolved in 20 mL of sterile distilled water. Besides, 20 g of maize grains were soaked in CFS (500 mg CFS/g of grains) for 8 h at room temperature after washing with distilled water. After that, the CFS-treated maize grains were shifted to the Petri plate. Similarly, 20 g of maize grains were soaked in MRS broth instead of CFS to be used as control and shifted to another Petri plate. A 5 mL of A. flavus (10 6 spores/mL) culture was spread over grains in both plates and examined for seven days, during incubation at 30˚C.

Fourier Transform Infrared Spectroscopy (FT-IR)
For the FT-IR analysis, 10 g of untreated maize grains (T1 = without any treatment) and treated maize grains (T2 = MRS broth + A. flavus (Control), and T3 = CFS + A. flavus) were milled into refined powder. The FT-IR spectrum (Tensor II, Bruker, Billerica, MA, USA) of powdered maize grain samples were recorded within the range of 4000-400 cm −1 absorbance mode to observe the nutritional contents of untreated and treated maize grains [35].

Total phenolic, flavonoid contents, antioxidant activity of treated and untreated maize grains
A 10 g of untreated (T1) and treated (T2 and T3) powdered maize grain samples were dissolved in 30 mL ethyl acetate and the supernatant was collected. After extraction, the solvent was evaporated by a rotary evaporator (R-210, Buchi, Flawil, Switzerland) at 40˚C. Furthermore, 1 mL extract of each treatment was dissolved in absolute ethanol [1:1 (v/v)] to be used for the determination of total phenolic and flavonoid contents, and antioxidant activity [36].

Total phenolic contents (TPC)
Folin-Ciocalteu method was used to determine phenolic contents spectrophotometrically [37]. Briefly, 0.5 mL of 10% Folin-Ciocalteu reagent was added in the test tubes containing 1 mL extracts or treatment solution of maize grains (T1, T2, and T3). Absolute ethanol was used as blank. The aqueous solution of sodium carbonate (2.5 mL of 700 mM) was added to each reaction mixture. The reaction tubes were vortexed, covered and incubated for 2 h at room temperature. The absorbance of each reaction mixture was measured against blank at 765 nm [37]. The measurements were compared to the standard curve of Gallic acid solution, and total phenolics were expressed as milligram of Gallic acid equivalent (GAE) per mL of (T1, T2, and T3) extract (mg GAE/mL of extract).

Total flavonoid contents (TFC)
Aluminum chloride colorimetric method was used to determine total flavonoid contents in untreated (T1) and treated extracts (T2 and T3) spectrophotometrically [38]. Briefly, 0.4 mL of 5% sodium nitrate (w/v) was added into 1 mL ethanol extracts (T1, T2, and T3) in test tubes. Absolute ethanol was used as a blank. After 5 min, 0.6 mL of 10% AlCl 3 .6H 2 O (w/v) and 1 M NaOH were added to the mixture, mixed well and absorbance was measured at 510 nm against the blank. The measurements were compared to standard curve of quercetin solution, and total flavonoid contents (TFC) were determined as mg of quercetin per mL of (T1, T2, and T3) extract (mg quercetin/mL of extract) [38].

Antioxidant activity by DPPH assay
The antioxidant potential of treatment solutions (T1, T2 and T3) was evaluated using 2, 2-diphenyl-1-picrylhydrazyl (DPPH) scavenging assay. This method is widely used due to its simplicity, reproducibility, and stability. The method is based on reduced DPPH which, is a stable free radical, having odd electrons and it gives maximum absorbance at 517 nm (purple color) [39]. Total, 30 μL each of untreated and treated maize grain extracts were added to 1 mL of DPPH ethanol solution (0.2 mM). The reaction mixture was incubated for 30 min at room temperature. When antioxidants react with DPPH, it is reduced by capturing electrons to form DPPH-H, resulting in decolorization (yellow color) and decrease in absorbance. Besides, 1 mL DPPH ethanolic solution was used as a control and the absorbance of samples and control was determined at 517 nm [39].
Percentage inhibition (%) of extracts was calculated using the formula given below: Where, A 0 : Absorbance of the DPPH solution; A 1 : Absorbance of each maize grain extract (T1, T2, and T3).

Aflatoxins analysis
Powdered maize grain samples (T1, T2, and T3) were added to 60 mL of methanol : water (8:2) solution and shaken for 30 min at room temperature, under dark conditions. The extracts were filtered with Whatman filter paper and 10 mL of these extracted filtrates were diluted with 70 mL of phosphate buffer saline (PBS). The samples were again filtered through a fiberglass filter for the separation of maize pigments. Immunoaffinity columns (Aflatest, R-biopharm, France) were used for sample cleaning up before high-performance liquid chromatography (HPLC) (FINNIGAN SURVEYOR, Thermo electron Corporation, USA

Statistical analysis
Inhibition zone was calculated as the mean ± standard deviation of three replicates (n = 3) and statistical analysis was performed by applying ANOVA, using GraphPad Prism software version 5.0 for Windows (GraphPad Software, La Jolla California, USA). Furthermore, Tukey's test was applied to find out the significance between total phenolic, flavonoid contents, and antioxidant treatments (p � 0.05), and all the data was presented as mean ± standard error.

In vitro antifungal potential of Loig. coryniformis BCH-4 CFS and its stability after various treatments
The CFS of Loig. coryniformis BCH-4 showed 16.33 ± 0.57 mm inhibition zone against A. flavus compared to negative control (no zone of inhibition) at 30˚C after 48 h ( Fig 1A). Besides, the CFS was found to be heat resistant because it did not lose its antifungal activity (16.56 ± 1.56 mm zone of inhibition) after heat treatment against A. flavus growth. However, the antifungal activity was decreased after treatment with proteinase K (10.66 ± 0.47 mm zone of inhibition) and pepsin (3.44 ± 0.56 mm zone of inhibition), suggesting the presence of proteinaceous compounds in CFS. Moreover, no inhibitory effect was observed at neutral pH which indicated that most of antifungal compounds, present in the CFS of Loig. coryniformis BCH-4, were pH dependant. Similarly, no inhibitory zone was observed after treatment with catalase. The results of all treatments were compared with negative control (MRS broth and distilled water), as shown in Fig 1B.

FT-IR analysis of maize grains
The FT-IR spectra of T1 (untreated maize grains), T2 (MRS broth + A. flavus treated maize grains), and T3 (CFS + A. flavus treated maize grains) were interpreted within the spectral region of 4000-400 cm −1 . The absorption bands that appeared in the range of 3500-3000

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cm −1 are assigned to be-OH and NH stretching [42] and the absorption band in the region of 2922 cm −1 can be assigned to be the -CH stretching. Absorption bands 1635 cm −1 and 1540 cm −1 showed the presence of carbonyl stretching (C = O) [35] while absorbance at 1043 cm -1 indicated the presence of C-O-C, C-C and C-O functional groups [42,43]. In the spectra, the-OH, NH, C = O stretching indicated the presence of peptides linkage (protein contents), CH stretching showed the presence of fatty acid contents while C-O-C, C-C and C-O stretching indicated the presence of carbohydrate contents. The spectra of T1, T2, and T3 maize grains showed similar bands but the patterns and intensities were found to be different (Fig 3). They indicated that nutritional contents (proteins, lipids, and carbohydrates) were decreased in T2 maize grains and affected by the A. flavus infestation. But in T3, the absorbance of maize grains was increased compared to T1 and T2 grains due to the addition of CFS.

Total phenolic and flavonoid contents in untreated and treated maize grains
Total phenolic contents were significantly improved in T3 (104.93 mg GAE/mL of extract) compared to T1 (95.63 mg GAE/mL of extract), while decreased total phenolic contents (87.62 mg GAE/mL of extract) were observed in T2 compared to T1 (Fig 4A). It was also noted that the total flavonoid contents were also significantly improved in T3 (441.15 mg quercetin/mL

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Detoxification of Zea mays L. to save its nutritional value of extract) as compared to T1 (326.28 mg quercetin/mL of extract) whereas, decreased flavonoid contents were observed in T2 (321.48 mg quercetin/mL of extract) compared to T1 ( Fig  4B). Moreover, the Tukey's multiple comparison test was applied to check which treatments means were significantly varying from each other. It was revealed that T1, T2 and T3 were significantly varied (p < 0.05) from each other (S1 Table).

DPPH scavenging assay of untreated and treated maize grains
Antioxidant activity of treatment solutions (T1, T2 and T3) was evaluated by DPPH scavenging assay. It depicted that T3 have significantly higher (84%) antioxidant contents as compared to T1 (72%). Furthermore, the antioxidant activity of T2 treatment solution was significantly decreased (66%) due to the presence of fungus in comparison with T1 and T3 treatment ( Fig  4C). The results of Tukey's multiple comparison test also indicated the significant difference (p < 0.05) between treatments T1, T2, and T3, because the p-value of T1 vs T2, T1 vs T3 and T2 vs T3 is less than the 0.05 (S1 Table).

Determination of aflatoxins in untreated and treated maize grains
The aflatoxins (AFB1 and AFB2) were not observed in T1 and T3 maize grains extracts, even after seven days of incubation while in T2 maize grains, the concentrations of AFB1 and AFB2 each were 487 and 16 ng/g respectively (S1 Fig), due to the infestation of aflatoxigenic A. flavus. A linear curve, in the range of 50-500 ppb of aflatoxins standards solutions, with coefficient of determination (R 2 ) of 0.9998, was obtained, using linear regression equation, based on the peak area of each peak, for the analysis of aflatoxins.

Determination of phenolics / organic acids in CFS of Loig. coryniformis BCH-4
The  Table 1.

Discussion
The bioinfestation of plants and cereals is an important issue. Alternate methods for bioprotection of plants and cereals are being studied for sustainable agriculture worldwide. Many synthetic chemicals have been evaluated and used. However studies have shown health hazards and environmental issues regarding the use of such chemicals [44]. The bioactivity studies of probiotic microorganisms for applications, as natural preservative for food products and their eco-friendly effect on human health, remain to be of great interest [45]. The present study highlighted the bioactivity of Loig. coryniformis BCH-4 CFS by observing its antifungal potential against aflatoxigenic A. flavus strain, under different treatments ( Fig  1B), in comparison with untreated CFS against the toxigenic A. flavus strain, shown in Fig 1A. The culture free supernatant of Loig. coryniformis BCH-4 exerted growth resistance of A. flavus (Fig 1A). These results have been supported by similar findings for other species of lactic acid bacteria such as, L. plantarum BCH-1 [29], L. plantarum RS2 [46], and L. kefiri FR7 etc. [47].
The current results showed no loss in activity after heat treatment at 100˚C for 20 min which suggested that the antifungal compounds, present in CFS, are heat resistant. Similar findings have also been reported by Nayyeri et al. [48] that heat treatment did not influence the antifungal activity of CFS of L. plantarum. However, the activity of Loig. coryniformis BCH-4 CFS decreased against A. flavus after treatment with proteinase K and pepsin, suggesting the presence of proteinaceous antifungal compounds present in the CFS of Loig. coryniformis BCH-4 [33]. Comparably, after neutralizing the pH of CFS, no zone of inhibition was

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Detoxification of Zea mays L. to save its nutritional value observed against A. flavus. These results are consistent with Wang et al. [49] who studied antifungal properties of Lactobacillus isolates from kumis against Penicillium roqueforti and A. niger. Their findings showed that an increase in pH (from 3.8 up to 7) ultimately reduced the size of inhibition zone. Therefore, antifungal activity of LAB is based on several mechanisms such as organic acids production and hence pH variation or pH-dependent antifungal proteins [33]. Likewise, during the treatment of CFS with catalase, no inhibitory effect was evaluated against A. flavus; this verified the involvement of hydrogen peroxide as antifungal compound which was converted into oxygen and water by this enzyme [33]. Based on the finding of antifungal activity, CFS of Loig. coryniformis BCH-4 was employed for bioprotection of post-harvested maize grains, infected with A. flavus (Fig 2). The bioprotection effect in the current study could be attributed to various bioactive compounds, present in Loig. coryniformis BCH-4, namely organic acids, fatty acids, phenolic compounds, proteinaceous compounds etc. mainly known for their antifungal potential [21,50,51]. In our previous work, the organic acids; pyruvic acid, lactic acid, citric acid, malic acid, succinic acid, and malonic acid have been reported in Loig. coryniformis BCH-4, for their potent antifungal effects against A. flavus and A. fumigatus [31]. Other Lactobacillus species such as L. casei, L. rhamnosus, L. plantarum, L. paracasei and L. curvatus also produced lactic acid, acetic acids, formic, citric, succinic and glutamic acids [52]. Other kind of compounds namely, cyclic dipeptides cis-cyclo (L-Val-L-Pro), cis-cyclo (L-Phe-L-Pro), cyclo(Leu-Leu) and cyclo(L-Leu-L-Pro), produced by L. plantarum and Loig. coryniformis, have also been reported for antifungal activity against Ganoderma boninense and A. flavus [25,53,54]. We have also reported previously 12-hydroxydodecanoic acid, a fatty acid, having antifungal activity against A. flavus and A. fumigatus in Bukhari et al. [29]. Other four hydroxy fatty acids; 3-hydroxydecanoic acid, 3-hydroxy-5-cis-dodecanoic acid, 3-hydroxydodecanoic acid, and hydroxytetradecanoic acid from L. plantarum milab14 have also been reported for their antifungal activities [55]. The fungal inhibitory mechanism of Loig. coryniformis BCH-4 CFS includes membrane destabilization, proton gradient interference, enzyme inhibition, and creation of reactive oxygen species [26,56].
The FT-IR analysis (Fig 3) indicated the effect of A. flavus on basic macronutrients of maize grains like proteins, fats and carbohydrates, by observing their relative intensities of characteristic absorption peaks. The decreased intensities in T2 treatment of subject nutrients might be due to fungal breakdown of such nutritional components into smaller fragments that were further metabolized [57]. The complex protein molecules are also utilized for obtaining energy, by respiration and to synthesize hyphal wall in fungi [11,58]. In T3 treatment, the CFS of Loig. coryniformis improved the nutritional contents of maize grains and protected them from fungal attack, presumably due to the addition of CFS, that is a mixture of phenolic compounds, fatty acids, organic acids, and peptides etc. [59,60].
More precisely, the culture supernatant of Loig. coryniformis BCH-4 not only guarded against A. flavus infestation but also augmented with improved antioxidant contents of T3 treated maize grains (T3: CFS + A. flavus). The results revealed a significant improvement (p � 0.05) in total phenolic contents (104.93 mg GAE/mL of extract), total flavonoid contents (441.15 mg quercetin/mL of extract), and total antioxidant activity (84%) (Fig 4) of T3 treatment in comparison with T1 (untreated) and T2 (MRS broth + A. flavus treated). Similar findings of bioprotection have previously been reported for L. plantarum in soybeans and maize grains, against A. flavus [25,34,40]. Despite this, the present study first time described the significant improvement of antifungal potential in CFS treated T3 maize grains by improving its functional antioxidant contents. This finding was supported by our previous study [31] that the CFS of Loig. coryniformis BCH-4 had potent antioxidant potential by observing its DPPH scavenging activity. Moreover, other species of Lactobacillus (L. plantarum, L. helveticus) also had reported potent antioxidant activity [61]. The increased antioxidant contents, in T3 maize grains, was justified due to the addition of Loig. coryniformis BCH-4 CFS, a mixture of metabolites, including phenolic compounds [59,62]. Besides, in T2 treatment solution, the antioxidant contents (phenolics and flavonoids) and antioxidant activity were significantly (p � 0.05) decreased as compared to T1 and T3. It is believed that the observed decrease was due to the action of fungal polyphenol oxidases that catalyzes the oxidation of many antioxidant compounds including phenolics and flavonoids into quinones [63]. The process of oxidation badly affects the food components including degradation of many bioactive food compounds such as phenolics and flavonoids, along with loss of its nutritional values [14].
Moreover, aflatoxins analysis revealed a complete inhibitory production of AFB1 and AFB2 in T3 treatment, due to the presence of various bioactive compounds, present in the CFS of Loig. coryniformis BCH-4, in comparison with T2, having aflatoxins (AFB1 and AFB2) level of 487 and 16 ng/g respectively (S1 Fig). The results suggested that metabolic products of Loig. coryniformis BCH-4, based on their ability to decrease the growth of aflatoxigenic A. flavus and biosynthesis of aflatoxins, could be considered as a good agent for bioprotection of maize grains. Previously Nazareth et al. [34] has reported that L. plantarum CECT 749 CFS reduced the AFB1 level (6.9 ng/g) in maize as compared to the control group (278.4 ng/g). Moreover, some other previous studies have also shown the potential inhibition of A. flavus aflatoxins (AFB1) production by L. casei and L. plantarum in maize [34,40,64] while the current study describes the inhibition of A. flavus aflatoxins, using Loig. coryniformis BCH-4 CFS. The current finding was also supported by several other studies like L. plantarum AF1 that inhibit the growth and aflatoxins production of A. flavus in soybeans [25]. In another study, Kachouri et al. [65] found L. plantarum to be effective against A. flavus in olives by reducing AFB1 levels.
The HPLC analysis of Loig. coryniformis BCH-4 CFS showed the presence of chlorogenic acid, p-coumeric acid, 4-hydroxybenzoic acid (HB acid), caffeic acid, sinapic acid, salicylic acid, and benzoic acid (S2 Fig). The presence of these phenolic acids in CFS ultimately revealed an increase in total phenolic contents and antioxidant activity of T3, which can be directly augmented with bioprotection of maize grains. Nazareth et al. [34] identified phenolic acids such as Gallic acid, protocatechuic, chlorogenic acid, vanillin, sinapic acid, salicylic acid, and p-coumaric acid in L. plantarum CFS and documented for potent antifungal activity against A. flavus and Fusarium verticillioides. These identified phenolic acids have previously been reported as antifungal compounds [34].
The chlorogenic acid and p-coumaric acid were present in high concentrations, which have been previously reported for disrupting the fungal cell membrane structure [66,67]. Overall, the presence of phenolics / organic acids in CFS of Loig. coryniformis, improved the nutritional contents of maize grains and also contributed to increase the antifungal potential along with other bioactive metabolites such as organic acids, proteins, peptides, fatty acids etc., that are extensively considered as antifungal compounds [20,27].

Conclusions
The present work explored the antiaflatoxigenic potential of Loig. coryniformis BCH-4. The results highlighted no production of aflatoxins in CFS treated maize grains even after seven days of infestation with A. flavus. Besides, this treatment also improved the antioxidant contents and antioxidant activity of maize grains which might be due to the presence of various phenolic acids (chlorogenic acid, p-coumaric acid, 4-hydroxybenzoic acid, caffeic acid, sinapic acid, salicylic acid, and benzoic acid) in the CFS of Loig. coryniformis BCH-4. To the best of our knowledge these phenolic acids have not been reported earlier in this bacterium, therefore, provide conclusive evidence as biopreservative, by enhancing nutritional and functional antioxidant values of Zea Mays L. The results also provide new insights for the biotechnological employment of such probiotic bacteria as bioprotectant for other cereal grains in future.